1. Field of the Invention
The present invention relates to biologically active substance-secreting hybrid gel. More particularly, the present invention relates to a new hybrid gel which secrets a biologically active substance, and is useful as an external-use prescription such as an artificial skin used in the treatment of various difficult-to-cure diseases requiring long-term and continuous medication of physiological active substances in order to maintain biological functions.
2. Description of Related Art
Three methods are available to cure diseases which are caused by the loss or decrease in the functions of human cells for any reason. Namely, the lost or decreased functions are compensated for by
1) drug medication,
2) transplantation of organs/tissues or cells, or
3) gene therapy
Insulin dependent diabetes mellitus, for example, is caused by the destruction of the xcex2-cells, which produce insulin having the function of reducing the blood sugar level, and which occur in the islets of Langerhans of the pancreas. Patients who suffer from insulin dependent diabetes mellitus have a high blood sugar level, and as a result, the concentration of sugar in their urine increases. When the blood sugar level remains high, the functions of various human cells are damaged, causing serious complications.
It is thus necessary to externally dispense insulin and control the blood sugar level in order to cure the insulin dependent diabetes mellitus patients. Insulin dependent diabetics must have insulin doses administered several times every day for life. This is a serious physical and mental agony to the patient, and in addition, self-medication always involve risk of life because of possible mis-dispensing.
One of alternative methods to self-medication of insulin in transplantation of the pancreas or the islets of Langerhans (K. Kubota and Y. Idezuki, Nippon Rinsho: in Japanese, 48:1052, 1990). However, this treatment has a number of problems, such as, for example, few donors, difficulty of controlling immunorejection caused by the transplanted pancreas or the tissues, complicated surgical operations for transplantation requiring techniques of a high level, and hazards involved in the operation.
Gene therapy is one of the most exciting medical technique to being employed solve the above problems, and various gene therapies are being clinically tested for treating patients having serious diseases in the United States and other countries in the 1990s (N. K. Summers, Biotechnology 12;42, 1994). A method of treating diabetes based on the above technique has been proposed (R. F. Selden et al, The New England Journal of Medicine, 317(17): 1067, 1987). In this particular method, and insulin gene is introduced into culture cells, and the cells are transplanted to the body of the patient in order to assure continuous secretion of insulin produced by the introduced gene. This method has a number of problems such as, for example, difficulty of controlling secretion of insulin from the transplanted insulin producing cells and inability of removing the transplanted cells later from the body. It is generally known that the gene therapy is a promising and advanced medical technique for not only insulin dependent diabetes mellitus and genetic diseases such as serious immune deficiency diseases but also cancer, AIDS, and other hard-to-cure diseases. For this reason, many approaches have been proposed and gene therapy is actually being conducted in practical clinical cases. Most of these gene therapies use retrovirus-derived vectors to introduce genes into the cells utilizing cell infection of the virus.
This technique to use retrovirus-derived vectors has the drawback that the effectiveness of gene introduction depends on the affinity of the virus to the cells, and there is a possibility that the deactivated virus vectors will transform into wild retroviruses. In addition, conventional gene therapy generally has a problem in there being difficulty of controlling the introduced genes externally.
The present invention intends to provide a new art of transplanting cells containing a gene which encodes biologically active substance into the skin and controlling the expression of the gene externally. More specifically, the present invention intends to solve the problems of the prior art by transplanting biologically active substance-producing cells to the skin of a human body as a hybrid gel (cell-incorporating gel).
The present invention provides a biologically active substance-secreting hybrid gel, which consists of biologically active substance-producing cells and a biopolymeric gel.
In the case of said hybrid gel, a preferable embodiment is that the biologically active substance-producing cells are enclosed in or laminated on the biopolymeric gel, or laminated on the biopolymeric gel enclosing the biologically active substance-producing cells.
The present invention also provides a biologically active substance-secreting hybrid gel, which consists of biologically active substance-producing cells, animal skin cells and biopolymeric gel.
In the case of said hybrid gel, a preferable embodiment is that the animal skin cells are laminated on the biopolymeric gel enclosing the biologically active substance-producing cells; the biologically active substance-producing cells are laminated on the biopolymeric gel enclosing the animal skin cells; the animal skin cells and the biologically active substance-producing cells are laminated on the biopolymeric gel; or the animal skin cells or the biologically active substance-producing cells are laminated on the biopolymeric gel enclosing the animal skin cells and the biologically active substance-producing cells.
Furthermore, in the case of the biologically active substance-producing cells being enclosed in the biopolymeric gel, said cells are enclosed together with meshy material or porous membrane.
In the present invention, moreover, the biologically active substance-producing cells may be skin cells (i.e., skin fibroblasts or skin epidermal cells) which contain an expression vector recombinant with a DNA sequence encoding the biologically active substance such as insulin. And, said expression vector may be plasmid vector pBMG-neo-ins possessing insulin CDNA and neomycin resistance gene, or plasmid vector pRIS-proins-Ifur-IIfur-B10D which possesses mutant insulin gene expressing stable insulin by the action of furin.
According to the present invention, it will be possible to develop a gene therapy by skin transplantation allowing stable drug medication for a long time; alleviating pains of the patients; and allowing fine adjustment of the dosage and control of gene externally without using retrovirus-derived vectors that tend to invoke the risk of mutation to wild types as in the conventional technique.
The cells used in the invention are enclosed in or laminated on the biopolymeric gel of the present invention and produce a biologically native substance necessary for or deficient in the body, and the substance is continually secreted into the body. The production of biologically active substance is increased when meshy material or porous membrace, etc. are enclosed in the gel together with the biologically active substance producing cells. Thus the hybrid gel of the present invention can be effectively used as, for example, an external-use prescription such as an artificial skin. The gene expressing biologically active substance is introduced to the cells by, for example, plasmid vector, and thus, unlike conventional gene therapy, no risk of conversion into wild retro-viruses owing to the retrovirus-derived vectors is involved. In addition, the introduced gene can be easily controlled externally because the gene-containing cells are transplanted to the skin.
The following functions are available in concrete:
1) After transplantation, the biologically active substance is dispensed for a long time stably without the knowledge of the patient. This dramatically reduces the physical and mental agony of the patient subject to repeated medication in the conventional treatment.
2) A very simple surgery is used to transplant or remove the hybrid gel of the present invention from the skin. For this reason, the quantity of artificial skin to be transplanted can be adjusted at any time freely while watching the process condition of the treatment, It is thus easy to determine optimum conditions for treatment.
3) The rate of substance secretion from the cells in or on the gel can be controlled by means of inducible promoters to control expression of the DNA sequence encoding the biologically active substance and various induction stimuli (hormones, heavy metals, temperature, etc.) applied to the transplanted artificial skin. This allows fine adjustment of the substance secretion rate.
4) The transplanted cells are enclosed in or on the gel and thus are hardly affected by immunorejection of the patient. It is thus possible to decrease the quantity of immunosuppressants generally used in the transplantation of tissues in the conventional technique. The risk of side effects owing to the use of immunosuppressants is thus greatly reduced. Of course there is no problem of immunorejection if the cells of the patient himself are used in the gene therapy because such therapy is a self-transplantation.
5) Simple operation without the need of the patient""s hospitalization is safe and free from the risk incurred in conventional treatment. Because this is a transplantation to the skin, the condition of transplantation is visible externally at all times. The transplanted artificial skin can be removed when necessary.
Various biologically active substance-producing cells can be used in the present invention for incorporating expression vector with gene therefor in the cells. For example, insulin-producing cells can be prepared by transfecting placmid vector pBMG-neo-ins possessing cDNA of insulin and neomycin resistance gene (selection marker) to animal cells using a known method. Another method is to transfect plasmid vector pRIS-proins-Ifur-IIfur-B10D into animal cells. This plasmid vector contains mutant insulin gene that convert proinsulin expressed from the gene into insulin by the action of furin and by the substitution of the 10th amino acid in the insulin chain B.
The gel to accommodate the biologically active substance-producing cells may be prepared from, for example, collagen, fibrin, agarose, etc. by using known methods. For example, the hybrid gel containing cells with insulin genes therein may be prepared and used as artificial skin for curing diabetics in the following manner:
(1) Pieces of skin of an experimental animal are collected. Epidermal cells and fibroblasts, two major constituent cells of skin, are separated from the skin samples and cultured.
(2) Expression vector containing insulin gene is transduced into these cells to derive insulin-secreting cell lines.
(3) Hybrid type artificial skin with the insulin-secreting function is constructed from these cell lines using collagen gels, etc.
(4) The insulin-secreting hybrid type artificial skin is transplanted.
To be more specific, the hybrid gel secreting biologically active substance of the present invention can be manufactured in accordance with the method of Asaga et al (H. Asaga et al, Experimental Cell Research, 193: 167, 1991) as follows:
Quadruple concentrated medium of cell culture, serum, purified water, and, for example, collagen (0.5% solution) are mixed in the ratio of 2.5:1:2.5:4 according to the required quantity while cooling the mixture with ice. An aqueous solution of 1N sodium hydroxide is mixed dropwise into the mixture to adjust to pH 7.4. The mixture is separately injected into hydrophobic plastic laboratory dishes of 35 mm in diameter, 2 ml in each dish. The dishes are immediately transferred to a 37xc2x0 C. thermostat. The collagen solidifies in several minutes to produce gel. Biologically active substance-producing cells are mixed into the above mixture just before collagen solidifies in order to enclose the cells in the gel.
To allow meshy material or porous membrane to coexist in the gel, one needs only to mix these in the above solution together with the biologically active substance-producing cells.
Commercially available culture solutions, serum and collagen can be used in the present invention.
It is effective to give an appropriate strength to the collagen gels to facilitate transplantation of the product to the skin. An appropriate strength can be given to the gel by, for example, mixing an appropriate number of skin-derived fibroblasts according to the method of Bell et al (E. Bell et al, Proceedings of the National Academy of Sciences, 76(3): 1274, 1979). An appropriate strength can be given to the gel as a result of contraction of the gel owing to the fibroblasts. Skin-derived fibroblasts can be obtained, for example, by culturing a small portion of skin collected from the patient according to the primary explant technique (R. I. Freshrey, Culture of Animal Cells, Alan R. Liss, Inc., New York, 1987).
It is also effective to make the gel surface active to ensure good attachment to the skin by overlaying by culture skin-derived epidermal cells on the gel before they are transplanted to the skin.
Skin-derived epidermal cells to be overlaid on the gel may be obtained by culturing epidermal cells obtained from the skin of the patient himself in the same way as described for the fibroblasts using the method of, for example, Green et al (H. Green et al, Proceedings of the National Academy of Sciences 76: 5665, 1979).
It goes without saying that the present invention is effective also when the gel is transplanted subcutaneously without overlaying epidermal cells.
Practically, many forms are of the invention available.